Reference is made to Patent Specifications GB 2339900 (and corresponding PCT specification WO 00/05569), GB 2339901, and GB 23515555, whose disclosures are herein incorporated by reference, which describe an epifluorescence imaging system designed to measure the flourescence emitted from the bottom of an array of samples when they are irradiated from beneath by excitation light. FIG. 1, taken from these Applications, shows the use of an array of typically 8×12 trifurcated fibre optic bundles arrayed in a presentation plate on which the sample plate is placed. One sub-set of fibres within a bundle is used to bring in the excitation light, and two other sub-sets of fibres in each bundle collect emitted fluorescent light into two separate filter channels to enable simultaneous measurement of dual wavelengths. After the filters the light is transmitted through single fibres, 2×96 of them, to a fibre optic input face of a CCD camera. The arrangement of optical fibre bundles at the filters is shown in FIG. 2, and of the optical fibres at the input to the CCD camera is show in FIG. 3. FIG. 4 shows how the different categories of fibres are arranged in a hexagonal pattern within a bundle.
Typically an image can be taken every second, and repeated at approximately one-second intervals.
This system has proved to be an efficient, highly sensitive measurer of fluorescence in dual wavelengths, and the ability to measure dual wavelengths simultaneously is of great value for certain assays, such as FRET assays, where a change in the ratio of the signal strengths in the two wavelengths is an indicator of chemical binding or some other attribute of the assay.
This 96-channel system can be used to measure 96-well microtitre plates, or by stepping the microtitre plate over the presentation plate, larger assay presentations such as 384, 864, 1536, 3465 . . . , well plates (i.e. having and n2*96 pattern, where n is an integer).
This system is not optimised for the measurement of luminescence for which no excitation is needed, and in which a sample emits light by virtue of a chemical reaction occurring when reagents are interacting in the sample well.
Thus, for fluorescence, the fibre bundle is stood off below the well since light has to illuminate the sample evenly and receive fluorescence light with uniform efficiency from all parts of the well. This still applies even if the sample is a cellular layer on the base of the well.
Therefore although this epi-fluorescence system can be used to measure luminescence, it is not optimal, and is generally sensitive enough only for the brightest types of samples and assays. Examples of such assays include detection using luciferase enzyme; calcium detection, for example in cells, linked to the enzyme aequorin, which produces a flash of light during the reaction; and alkaline phosphatase linked assays using enhanced chemiluminescence substrates.